PROJECT SUMMARY Ubiquitination is a post-translational modification of proteins that is required for many cellular processes, including protein degradation by the proteasome, and requires the sequential action of three enzymes called E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin-ligase). While there is a single E1 enzyme and ~40 E2 enzymes, there are over 600 E3 enzymes in humans, with the RING (Really Interesting Novel Gene) family of E3 ubiquitin ligases being the most abundant. The RING E3 ubiquitin ligases recognize specific substrate proteins and interact with the ubiquitin-charged E2 enzymes to transfer the ubiquitin to the substrate. Recently, this pathway has been utilized to target physiologically relevant proteins for degradation using proteolysis-targeting chimeric (PROTAC) small molecules that bind to both an E3 ubiquitin ligase and a target protein. In the R61 component of this NIH grant proposal, we have chosen to validate the fertility essential functions of four evolutionarily-conserved testis-enriched ubiquitin-proteasome pathway proteins using a CRISPR/Cas9 knockout mouse strategy. Using CRISPR/Cas9, we have created mutations in all four of these genes, and preliminary data has revealed that at least one of these genes is required for male fertility. Our next step is to identify small molecules that bind these proteins using DNA-Encoded Chemistry Technology (DEC-Tec), in which each protein will be screened in vitro against our over 2.8 BILLION molecule collection. DEC-Tec is an economical process to rapidly uncover lead molecules, which will be tested directly for their inhibitory action or linked with other contraceptives to form PROTACs for targeted degradation of other spermatogenic-specific contraceptive targets. In the R33 component of this NIH grant proposal, we will use medicinal chemistry to optimize inhibitors for direct contraceptive action and create PROTACS that bind these E3 ubiquitin protein ligases and known contraceptives. Our R61/R33 proposal is an innovative, discovery- based, and integrated strategy to use mouse genetics to define the essential roles of these four testis-enriched ubiquitination-proteasome pathway proteins, to use DEC-Tec to rapidly identify small molecule binders, and to directly use these small molecules as non-hormonal contraceptives or to link them to other lead contraceptive compounds (as PROTACs) to direct the contraceptive target for degradation.